| Objective:To prepare uniform-sized Silybin loaded poly (lactic-co-glycolic acid)(PLGA) microspheres and study its influencing factors. Study their physical and chemical properties, release characteristics in vitro and their effect on non-alcoholic fatty liver in HepG2human hepatoma carcinoma cells. To establish experimental basis for Silybin PLGA microspheres’vivo studies in animals and support more Chinese medicine or natural medicine effective component (group) prepared as microspheres drug delivery system.Methods:To establish a RP-HPLC method for the determination of Silybin. Silybin as the model drug and PLGA as the carrier material, Silybin PLGA microspheres were prepared by premix membrane emulsification-solvent extraction/evaporation method. Both volume average particle size (D0.5) and span as the indicator, study their influencing factors by single-factor test, such as oil phase solvent, membrane pore size, transmembrane pressure, centrifugation time, solidifying liquid category and so on. Combined with the results of single factor test, screening three main factors, ratio of drug and carrier material, concentration of PVA, and volume ratio of oil phase to external water phase, the preparation conditions were optimized by orthogonal experiment. The quality of the microspheres were evaluating by studying their morphology, particle size and distribution, dispersion, drug loading, encapsulation efficiency, stability and other aspects. Immediate release method was used to study the drug release rule of Silybin PLGA microspheres. And examine the volume of the release medium to have an effect on the release rate of the microspheres. Long-chain mixture of free fatty acids (oleate and palmitate) was used to induce steatosis in HepG2cells. Lipid droplets were observed with oil red O stain and inverted electron microscope. The content of the TNF-a in culture supernate were tested by ELISA Kit.Results:The final optimum conditions were as follows:membrane pore size2.8μm, transmembrane pressure1.0MPa, centrifugation time20min, solidifying liquid category saline, ratio of drug and carrier material1:4, concentration of PVA3%, and volume ratio of oil phase to external water phase1:19. Prepared microspheres were round and with a smooth surface. The mean diameter was (2.634±0.35)μm. The span was (13.326±3.06). The average drug loading was (14.84±0.76) and entrapment efficiency was (56.16±3.77)%. In vitro release, along with the release medium volume’s increase, Silybin PLGA microspheres release rate speeded up. At last,40mL was chosen as the volume of release medium. Four batches silybin PLGA microspheres’ which prepared with the optimum conditions release behaviors are almost the same. After12h quick-release, they kept sustained release. The cell experiment showed that deposition of lipid in different concentration Silybin PLGA microspheres groups were significantly less than model group. The content of inflammatory factors TNF-a in culture supernate was lower than model group, with a significant difference (P<0.01).Conclusions:Evaluate the quality of silybin PLGA microspheres from their morphology, particle size and distribution, further dispersion, drug loading, encapsulation efficiency, stability, vitro release behavior and so on. The microspheres prepared with the optimal method had a uniform-sized, controlled particle size and stable quality. The premix membrane emulsification can be used to prepare the silybin PLGA microspheres which was an insoluble extracted from Chinese medicine Silybum marianum Gaertn. The result of release experiment suggested that there were a lot of free drug in microspheres system. A serious sudden release would be caused by them. At a later stage, try to improve the preparation. But it failed. Significantly Silybin PLGA microspheres can improve the fatty deposition in HepG2cells induce by long-chain mixture of free fatty acids and inhibit the secretion of TNF-a. |
PDF Full Text Request